Rate and capacity of hepatic microsomal ring-hydroxylation of phenol to hydroquinone and catechol in rainbow trout (Oncorhynchus mykiss)

Article abstract of DOI:10.1016/S0300-483X(02)00144-0

Rainbow trout (Oncorhynchus mykiss) liver microsomes were used to study the rate of ring-hydroxylation of phenol at 11 and 25°C by directly measuring the production of two potentially toxic metabolites, hydroquinone (HQ) and catechol (CAT). An HPLC method

Full text of DOI:10.1016/S0300-483X(02)00144-0

Toxicology 176 (2002) 77–90
www.elsevier.com/locate/toxicol
Rate and capacity of hepatic microsomal ring-hydroxylation
of phenol to hydroquinone and catechol in rainbow trout
(Oncorhynchus mykiss)
Richard C. Kolanczyk *, Patricia K. Schmieder
Mid-Continent Ecology Di6ision, US En6ironmental Protection Agency, 6201 Congdon Bl6d, Duluth, MN 55804, USA
Received 24 January 2002; received in revised form 3 April 2002; accepted 4 April 2002
Abstract
Rainbow trout (Oncorhynchus mykiss) liver microsomes were used to study the rate of ring-hydroxylation of phenol
at 11 and 25 °C by directly measuring the production of two potentially toxic metabolites, hydroquinone (HQ) and
catechol (CAT). An HPLC method with integrated ultraviolet and electrochemical detection was used for metabolite
identification and quantification at low (pmol) formation rates found in fish. The Michaelis–Menten saturation
kinetics for the production of HQ and CAT over a range of phenol concentrations were determined at trout
physiological pH. The apparent Km’s for the production of HQ and CAT at 11 °C were 1491 and 1091 mM,
respectively, with Vmax’s of 552971 and 161915 pmol/min per mg protein. The kinetic parameters for HQ and
CAT at 25 °C were 2291 and 3293 mM (Km) and 17529175 and 940973 pmol/min per mg protein (Vmax),
respectively. The calculated increase in metabolic rate per 10 °C temperature rise (Q ) was 2.28 for HQ and 3.53 for
10
CAT production. These experiments assess the potential for metabolic bioactivation in fish through direct quantifica-
tion of putative reactive metabolites at the low, but toxicologically significant, chemical concentrations found in
aquatic organisms. This work initiates a series of studies to compare activation pathway, rate, and capacity across fish
species, providing a basis for development of biologically-based dose response models in diverse species. © 2002
Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Phenol; Bioactivation; Electrochemical detection; Rainbow trout; Metabolism; Hydroquinone; Catechol
ture and industry in the US (Interagency Testing
1. Introduction
Committee, 1990), with annual production vol-
umes well in excess of one million pounds. The
Environmental Protection Agency’s Toxics Re-
lease Inventory (1990) indicates that each year
large quantities of phenolics are released to air,
water, and land. Metabolism of phenol is also of
substantial toxicological interest. The biotransfor-
mation of xenobiotics is generally considered
Phenol, substituted phenols, and chemicals that
can be metabolically converted to phenolic com-
pounds continue to be used extensively in agricul-
*
Corresponding author. Tel.: +1-218-529-5152; fax: +1-
2
18-529-5003.
E-mail address: kolanczyk.rick@epa.gov (R.C. Kolanczyk).
0
300-483X/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0300-483X(02)00144-0

7
8
R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
to be a means of chemical detoxification. Phase
I oxidation reactions for common substrates,
such as phenol (Daly et al., 1965; Irons and
Sahawata, 1985) serve to increase chemical po-
larity and provide substrate for Phase II conju-
gation reactions thus facilitating chemical
elimination, as demonstrated for both aquatic
and mammalian species (Mulder, 1982; Layiwola
et al., 1983; Nagel, 1983). However, it has also
been recognized that metabolic transformation
may result in more toxic chemical forms. For
instance, the conversion of benzene to phenol
may facilitate subsequent conversion to reactive
quinones such as hydroquinone (HQ) and ben-
zoquinone (BQ) (Medinsky et al., 1995).
mammalian liver microsomes, tissue ho-
mogenates, cell suspensions, liver slices, whole
organs, and using in vivo exposures (Lunte and
Kissinger, 1983; Sahawata and Neal, 1983;
Schlosser et al., 1993; Kenyon et al., 1995; Hoff-
man et al., 1999) with a myriad of analytical,
perfusion and microdialysis techniques (Lunte
and Kissinger, 1983; Scott et al., 1989; Davies
and Lunte, 1995). By comparison, there is rela-
tively little detailed information available regard-
ing potential bioactivation pathways, and
associated rates, that can be used to model the
disposition of potentially reactive metabolites of
phenol in aquatic organisms. Early reports of
phenol metabolism in fish centered on the iden-
tification of sulfate and glucuronide conjugates
with no evidence for HQ formation (Kobayashi
and Akitake, 1975; Layiwola and Linnecar,
Bioactivation reactions which have been stud-
ied to a considerable extent in mammals (see
Guengerich and Liebler, 1985; Anders, 1985;
Hinson et al., 1994 for examples) have also been
found to be important in aquatic species
1
981). Indirect evidence for the formation of HQ
in phenol exposed fish was shown by the iden-
tification of the conjugate HQ-sulfate (Nagel,
(
Varanasi and Stein, 1991; Dady et al., 1991;
Bradbury et al., 1993; Kolanczyk et al., 1999).
Comparisons between mammalian and aquatic
metabolic enzyme systems have been made for a
few standardized substrates (Gregus et al., 1983;
Funari et al., 1987). Yet, there exists relatively
little information on pathways and rates of
metabolic conversion to potentially more toxic
chemical forms, particularly with regard to
aquatic species. This lack of information ham-
pers the development of models predictive of
chemical toxicity for aquatic species, particularly
for compounds likely to be more toxic after
metabolic conversion than originally predicted
based on parent chemical mode of action (Rus-
som et al., 1997). Additionally, physiologically-
based kinetic models used to predict chemical
disposition in aquatic species currently work well
for chemicals not significantly metabolized
1
1
983; Nagel and Urich, 1983; Kasokat et al.,
987). More recently McKim et al. (1999) re-
ported the direct measurement of HQ in the
plasma and urine of rainbow trout exposed to
phenol in the water. This type of measurement
was only recently possible due to the application
of sensitive analytical techniques for detection of
low but relevant metabolite concentrations. The
in vivo data collected can serve as the basis for
the validation of fish physiologically-based
model for phenol and its’ metabolites. However,
in vitro metabolic pathway, rate and capacity
data is needed to provide independent parame-
terization of models prior to validation, and for
further extension of the modeling approach to
many more chemicals of diverse structure.
The current study was undertaken to identify
pathway (Phase I oxidative products) and for-
mation rate of enzymatic ring hydroxylation
metabolites of phenol in the rainbow trout, a
species commonly used for chemical uptake,
metabolism, disposition, and toxicity studies
(Franklin et al., 1980; Melancon and Lech, 1984;
McKim et al., 1987a,b; Buhler, 1995), and for
further development of fish physiologically-based
toxicokinetic models (Nichols et al., 1990; Lien
(
Nichols et al., 1990; Lien et al., 2001), but do
not typically predict production and disposition
of bioactivated metabolites. Incorporation of
metabolism into predictive models requires
knowledge of metabolic pathway (products),
rates of formation, and characterization of enzy-
matic capacity.
The metabolic conversion of phenol to qui-
none metabolites has been studied extensively in

R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
79
et al., 2001). Due to the relatively slow rate of
2.2. Standard and sample preparation and
handling
metabolic conversion seen in aquatic species rela-
tive to mammals (often 10–100-fold less) the use
of mM substrate concentration results in uM to
nM formation of product, necessitating the incor-
poration of highly sensitive analytical detection
Standards and samples required special precau-
tions to ensure stability. In all cases, cold solvents,
buffers and water were used to dissolve solid
compounds. Solutions were kept on ice and pro-
tected from the light to avoid degradation of
analytes (Kolanczyk et al., 1999). HQ, CAT and
BQ standard solutions were prepared daily in
acetonitrile:water (1:1).
(
Kolanczyk et al., 1999). However, orders of mag-
nitude greater sensitivity of fish to HQ than phe-
nol (DeGraeve et al., 1980) makes relatively low
rates of HQ production still toxicologically signifi-
cant. Study objectives were to (1) identify poten-
tially bioactive Phase 1 metabolites of hepatic
microsomal biotransformation of phenol in trout
using more sensitive analytical detection than pre-
viously applied; (2) characterize the linear forma-
tion rate and enzymatic biotransformation
capacity for each metabolite formed by utilizing
phenol concentrations necessary to achieve en-
zyme saturation; and (3) determine metabolic
product production at physiological temperature
relevant for this cold water species, as well as
room temperature, to serve as the basis for ex-
trapolation to other species. Additionally, forma-
tion of HQ through enzymatic ring hydroxylation
determined in the current effort was compared
with HQ production via O-dealkylation previ-
ously measured under identical conditions
2.3. Animals
Juvenile rainbow trout (Oncorhynchus mykiss)
(100–250 g) from Seven Pines Fish Hatchery
(Lewis, WI, USA) were held for several weeks in
flow-through 815 l tanks with sand filtered Lake
Superior water (4 l/min) at 11 °C. Trout were fed
commercial Silver Cup trout pellets from Nelson
and Sons Inc., (Murray, UT) three times a week
at a rate of 1.2% body weight per day, and held
under a 16-h light:8-h dark photoperiod.
2.4. Microsomal characterization
Liver microsomes were prepared from fasted
rainbow trout as previously described (Kolanczyk
et al. 1999). Due to the relatively small size of
fish used, and the yield of liver tissue per individ-
ual (generally 1–1.5% of total body weight),
it was necessary to pool animals to obtain ade-
quate tissue to complete the required incubations.
Livers from three trout were combined, without
regard to gender or size (100–250 g), for prepara-
tion of each of five microsomal samples. Previous
investigations in this laboratory have observed no
apparent differences in Phase 1 biotransformation
of phenol for immature rainbow trout regardless
of size and gender (unpublished observations).
Isolated microsomes were stored at −80 °C
for up to 6 months (Forlin and Andersson, 1985).
Each microsomal sample was characterized as to
total protein (Bradford, 1976), P450 content per
mg microsomal protein as measured by the
method of Estabrook et al. (1972) using an ex-
tinction coefficient of 0.1 mM/cm and 7-ethoxy-
resorufin-O-deethylase (EROD) activity by a
(
Kolanczyk et al., 1999).
2
. Materials and methods
2
.1. Chemicals
Substrate (phenol) and metabolite standards
(
HQ, catechol (CAT), BQ) were obtained from
Aldrich Chemical Company (Milwaukee, WI,
USA). Reducing equivalents, buffer components,
G-6-P dehydrogenase and 7-ethoxyresorufin were
purchased from Sigma Chemical Co. (St. Louis,
MO, and USA). Acetonitrile and methanol from
Burdick and Jackson (Muskegon, MI, USA) were
of analytical grade. Resorufin was obtained from
Pierce Chemical Company (Rockford, IL, USA).
Disodium
ethylenediaminetetraacetate
and
sodium dithionite were purchased from Fisher
Scientific (Eden Prairie, MN, USA).

8
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R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
modified method of Pohl and Fouts (1980) utiliz-
ing excitation and emission wavelengths of 530
and 585 nm, respectively. EROD reaction product
formed after 10 min at 11 °C was quantified
against a resorufin standard curve.
or reduction potentials for each analyte. Optimum
ECD potential for detection of HQ was E°= +
0.425 V [ox], for CAT was E°= +0.550 V [ox]
and for BQ was E°= +0.050 V [red] (range=10
nA; filter 0.1 Hz). ECD stability and instrument
performance were assessed on a daily basis. Dual
channel ECD was used for peak identification and
quantitation of HQ, CAT and BQ, compared
with standards. We have previously determined a
detection limit for HQ, BQ and CAT equivalent
to 0.02 uM (Kolanczyk et al., 1999). UV-diode
array detection was used to quantify levels of
phenol, and as additional confirmation of
metabolite identification.
2.5. Microsomal incubations
Incubations of five microsome samples per phe-
nol concentration for metabolite identification
and metabolic rate determination were conducted
in open microcentrifuge tubes containing the fol-
lowing constituents in a final volume of 500 ml: 25
ml 20 mM MgCl , 25 ml 10 mM G-6-P, 121 ml 13.5
2
mM NADP, 40 ml (5 U) G-6-P dehydrogenase, 50
ml microsomes (0.8–0.95 mg/ml protein), and var-
ious concentrations of phenol (0.70–80 mM) in
2.7. Data analysis
239 ul 0.1 M Trizma–HCl buffer (pH 8.0). Micro-
somes and cofactors were incubated for 5 min
prior to initiation of reaction by addition of sub-
strate. Incubations were conducted for 15 min in a
temperature controlled reciprocal shaker at 11 or
Measurements shown are the mean9standard
error of duplicate or triplicate observations made
using each of five microsomal preparations, at
each of ten substrate concentrations. Estimates of
apparent Km and Vmax (9S.E.) were done in
two ways. For each microsomal preparation a
saturation curve was fit to ten points, i.e. the
mean measured HQ or CAT production at each
substrate concentration, resulting in an apparent
Km and Vmax estimate for each preparation. A
mean Km and Vmax was then calculated from the
five individual rate constants, or the ‘mean of
individuals’ (Tables 1 and 2). A second calcula-
tion of an apparent Km and Vmax was done by
first obtaining the average production of HQ or
CAT across all five preparations measured at each
of ten phenol concentrations, then fitting a curve
to the points from which the ‘mean rate across
preparations’ were obtained (Tables 1 and 2). A
non-linear least squares regression program (EZ-
Fit™ version 5.03; Perrella Scientific; Amherst,
NH, USA) was used to generate parameter esti-
mations from untransformed kinetic data.
2
5 °C. Cold (4 °C) ZnSO 25% (0.05 ml) and
4
Ba(OH) saturated (0.05 ml) were added to stop
2
the reaction. Samples were then vortexed, stored
on ice for 5 min, and centrifuged 3 min at
18 200×g. Samples were placed back on ice for 5
min, and centrifuged 3 min at 18 200×g. Super-
natant was transferred to amber HPLC vials
equipped with inserts, maintained at 4 °C, and
analyzed immediately by HPLC to preserve sam-
ple integrity.
2.6. Metabolite identification and quantification
Analysis of microsomal incubation samples was
performed on a Beckman System Gold HPLC,
equipped with a refrigerated autosampler, diode
array UV detector, and a BAS CC-5 dual channel
LC-4C
Kolanczyk et al., 1999). Injections (20 ml) were
made onto a Shandon Hypersil ODS (C18) 5u
.6×250 mm column. An isocratic mobile phase
1 ml/min) consisting of 9.6% ACN and 0.1 M
electrochemical
detector
(ECD)
(
The Q -values of reaction rates were calculated
1
0
1 2
10/(T −T )
4
(
from the equation: Q =(K /K )
where,
10
1
2
K and K are velocity constants corresponding to
1
2
sodium acetate (pH 4.2) was employed. The ECD
featured a glassy carbon working electrode and
Ag/AgCl reference electrode, using hydrodynamic
voltammograms to determine optimum oxidation
the temperatures T and T .
1 2
Statistical comparisons between groups (n=9)
were performed using the unpaired t-test at P5
0.05.

R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
81
Table 2
3. Results
The fitted Michaelis–Menten kinetics for ring-hydroxylation in
juvenile rainbow trout microsomes over a range of phenol
concentrations (1–80 mM) at 25 °C
The metabolic products identified upon phenol
incubation with trout microsomes were the para-
and ortho-ring hydroxylation products, HQ and
CAT, respectively. The amount of CAT produced
was typically one-half to one-third less than the
amount of HQ formed. There was no BQ detected
in any of the incubations. Sensitive detection of
nanomolar quantities of HQ and CAT produced
from mM phenol substrate incubations was
achieved using HPLC separation with ECD opti-
mized through the use of hydrodynamic voltam-
mograms. Microsomal assay conditions were
optimized for pH, incubation time, cofactor and
Preparation
Km (mM)
Vmax
pmol/min per mg)
(
Hydroquinone
c1
c2
c3
c4
c5
17910
21913
22911
23912
25911
14159293
14979357
19639389
15449315
23439429
17529175
Mean of individual 2291
a
rates
Mean rate across
21911
17459347
preparationsb
Catechol
c1
Table 1
29919
26919
39919
28917
38916
8539236
7209219
11319267
10629267
9329185
940973
Michaelis–Menten kinetics for ring-hydroxylation in juvenile
rainbow trout microsomes over a range of phenol concentra-
tions (1–60 mM) at 11 °C
c2
c3
c4
c5
Preparation
Km (mM)
Vmax
pmol/min per mg)
Mean of individual 3293
(
a
rates
Mean rate across
31918
9319227
Hydroquinone
preparationsb
c1
1593
1194
1295
1595
1897
1491
480936
393939
586979
492953
8099118
552971
c2
Each preparation represents the pooled livers from three fish.
c3
a
Michaelis–Menten constants (mean9standard error) cal-
c4
culated from five Km and Vmax values resulting from the
saturation curve fitting of HQ or CAT production data at each
phenol concentration from five individual microsome prepara-
tions.
c5
Mean of individual
ratesa
Mean rate across
preparations
1595
575965
b
Km and Vmax resulting from the saturation curve fitting
b
of the average HQ or CAT production data at each phenol
Catechol
c1
concentration of five individual microsome preparations.
12910
996
1097
896
1295
1091
144935
112920
188936
198939
163922
161915
c2
c3
microsomal protein concentration. Optimum HQ
formation occurred at pH 8.0, 74% less was de-
tected at pH 7.4, and 33 and 99% less was seen at
pH 8.5 and 6.5, respectively (Fig. 1). CAT forma-
tion was also favored at pH 8.0. The pH optima
of 8.0 corresponded to the plasma pH for rainbow
trout. The rates of HQ and CAT production with
the concentration of cofactors utilized, were found
to be linear with respect to time up to 20 min. No
HQ or CAT formation was detected in absence of
phenol or one or more cofactors from the incuba-
tion mixture, thus verifying absence of endogenous
production of metabolites or compounds that may
interfere with detection of compounds of interest.
c4
c5
Mean of individual
ratesa
Mean rate across
preparations
1297
179930
b
Each preparation represents the pooled livers from three fish.
a
Michaelis–Menten constants (mean9standard error) cal-
culated from five Km and Vmax values resulting from the
saturation curve fitting of HQ or CAT production data at each
phenol concentration from five individual microsome prepara-
tions.
b
Km and Vmax resulting from the saturation curve fitting
of the average HQ or CAT production data at each phenol
concentration of five individual microsome preparations.

8
2
R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
The concentration of phenol freely dissolved in
microsomal incubations at 11 °C was suspect due
to previous observations of decreased solubility of
another substrate, 4-methoxyphenol, during low
temperature incubations (Kolanczyk et al., 1999).
The concentration of phenol freely dissolved, de-
termined by acetonitrile addition to microsomes
(
Kolanczyk et al., 1999), was equal to nominal
concentrations at 25 °C, i.e. 1–80 mM, but
7.297.5% less than nominal at 11 °C (data not
2
shown). Phenol concentrations were corrected for
this 27% loss prior to calculation of kinetic con-
stants at 11 °C, yielding a range of 0.73–58 mM.
No loss of either metabolite, HQ or CAT, was
observed during sample processing.
Incubations of trout liver microsomes with phe-
nol (0.73–80 mM) were conducted at the physio-
logically relevant temperature of 11 °C (Fig. 2)
and also at 25 °C (Fig. 3) for direct comparison
with other enzyme studies. At 11 °C, nearly linear
production of HQ was observed to 20 mM phenol
followed by apparent enzyme saturation with
measured maximum rates of 350–400 pmol/min
per mg protein for most microsome preparations.
However, HQ production of 650 pmol/min per
mg was measured for one preparation, illustrating
the potential for large variation in rates between
fish (Fig. 2A). Production of CAT at 11 °C fol-
lowed a similar trend with a linear increase up to
Fig. 2. Rates of simultaneous production of (A) HQ and (B)
CAT formation resulting from the incubation of phenol with
juvenile rainbow trout microsomes at 11 °C. Each symbol
represents the mean9S.E. for one of five different prepara-
tions of microsomes containing three fish livers each. Prepara-
tion c 1 (ꢀ), c2 (ꢁ), c3 (ꢂ), c4 (2), and c5 (ꢃ).
Lines represent a curve using average rate constants over five
microsome preparations.
1
1
5 mM phenol, above which a steady rate of
20–140 pmol/min per mg was observed for the
majority of microsome preparations (Fig. 2B). As
with HQ, at least one microsomal preparation
produced almost double the usual CAT rate.
Production of HQ and CAT at 25 °C followed
a similar pattern to that observed at the lower
temperature when considering relative rates, with
HQ\CAT, and inter-microsome variability (Fig.
3
A and B). However, much greater absolute rates
were measured with a usual rate of 1200 pmol HQ
per min per mg going up to 1800 pmol HQ per
min per mg for one preparation, and a usual rate
of 600 pmol CAT per min per mg extending as
high as 900 pmol CAT per min per mg for one
set.
Fig. 1. Effect of pH on the ring-hydroxylation of phenol to
HQ (ꢄ) and CAT (ꢀ).

R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
83
The Michaelis–Menten saturation kinetics for
the production of HQ and CAT via ring-hydroxy-
lation were evaluated. Estimates of apparent Km
and Vmax (9S.E.) were done in two ways to
demonstrate not only average rates but also the
wide variation measurable among fish. At 11 °C
the apparent Km for phenol hydroxylation to HQ
was 1491 and to CAT was 1091 mM, with
corresponding Vmax values of 552971 and
CAT, with Vmax=575965 pmol HQ per min
per mg protein and 179930 pmol CAT per min
per mg protein. While either method for calcula-
tion of mean Km and Vmax seems adequate,
determination of rate constants for individual mi-
crosomes provides additional information. While,
Km did not appear to be different across prepara-
tions, the highest Vmax for HQ production at
11 °C (809 pmol/min per mg protein), measured
for preparation c5 (Table 1), was twice that of
the lowest (393 pmol/min per mg protein), mea-
sured for preparation c2.
161915 pmol/min per mg protein, respectively,
calculated as the ‘mean of individual’ prepara-
tions (Table 1). The resulting kinetic constants
calculated as the ‘mean across preparations’ were:
Km=1595 mM for HQ, and 1297 mM for
Similar variations in reaction rates among mi-
crosomal preparations were noted at 25 °C. The
Km, calculated as ‘mean of individuals’ prepara-
tions were 2291 mM (HQ) and 3293 mM
(
CAT); the Vmax was 17529175 pmol HQ per
min per mg protein and 940973 pmol CAT per
min per mg protein (Table 2). As was noted with
the lower temperature, constants calculated as
‘
mean across preparations’ were similar to those
calculated as ‘mean of individuals’, with a Km of
1 mM for HQ and 31 mM for CAT, and Vmax
2
of 1745 pmol HQ per min per mg protein and 931
pmol CAT per min per mg protein, respectively
(
Table 2). Interestingly, the apparent Km esti-
mated for CAT at 25 °C was greater than the
corresponding Km for the HQ reaction at the
same temperature, a trend not observed when
metabolism was measured at the physiological
temperature for trout of 11 °C. Despite the lower
apparent enzyme affinity, the maximal rate of
CAT production increased more than expected in
comparison to rate increases with temperature
noted for HQ production. This was best demon-
strated by comparing calculated rate changes with
a 10 °C increase in temperature (i.e. Q ). The Q
10
10
for maximal production of HQ was 2.28 and for
CAT was 3.53, which were shown to be statisti-
cally different (PB0.0001) over the five prepara-
tions of microsomes. The Km, 1493 mM
(
mean9S.D.), for the formation of HQ at 11 °C
was shown to be statistically different (P=
.0036) than that at 25 °C, 2293 mM. A temper-
Fig. 3. Rates of simultaneous production of (A) HQ and (B)
CAT formation resulting from the incubation of phenol with
juvenile rainbow trout microsomes at 25 °C. Each symbol
represents the mean9S.E. for one of five different prepara-
tions of microsomes containing three fish livers each. Prepara-
tion c 1 (ꢀ), c2 (ꢁ), c3 (ꢂ), c4 (2), and c5 (ꢃ).
Lines represent a curve using average rate constants over five
microsome preparations.
0
ature effect (P=0.0002) was observed on the
Vmax for the formation of HQ at 11 °C (5529
59 pmol/min per mg) and 25 °C (17529392
1
pmol/min per mg). Statistical difference was also

8
4
R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
observed for CAT formation Km and Vmax (PB
validation of metabolism simulation models. A
better understanding of how metabolic pathway,
linear rate, and maximal capacity vary with chem-
ical structure is needed for more accurate predic-
tion of the potential for metabolic activation, with
associated increases in toxicity, as well as for
accurate prediction of metabolic detoxification
leading to decreased accumulation and reduced
toxicity. While many aquatic species have been
characterized as to content of multiple P450 iso-
forms (Buhler, 1995), relatively less is known
about rate of conversion of the wide variety of
chemical substrates aquatic species are potentially
exposed to, with the exception of a few well
characterized ‘standard’ substrates. Specific path-
way and rate information is needed to account for
not only parent chemical, but also disposition of
metabolites in physiologically-based toxicokinetic
models for fish, and ultimately for development of
linked physiologically-based kinetic models with
toxicodynamic models that would allow estima-
tion of a chemicals’ increased toxic potential
through accurate prediction of metabolic bioacti-
vation rate.
HQ was the major metabolite produced by
hepatic microsomal biotransformation of phenol
in trout. CAT was also found to be a significant
ring-hydroxylation product of phenol, although
produced in lesser amounts. Microsomal
metabolism of phenol measured in rodent species
resulted in HQ:CAT product ratios of 20:1 in rats
(Sahawata and Neal, 1983) and about 15:1 in
both rats and mice (Schlosser et al., 1993). This
study allowed the first estimation of HQ:CAT
product ratios for trout, which were 3.5:1 at
11 °C and 2:1 at 25 °C. These results indicate
there is greater relative ortho-hydroxylation rate
in fish compared with that observed in rodents,
although absolute rates of metabolism are lower
in aquatic species. At physiological temperatures
the rate of microsomal production of HQ from 1
mM phenol was 7020 pmol/min per mg in rats
(Sahawata and Neal, 1983), about 350 times more
than in trout at 19 pmol/min per mg. While
biotransformation of mM concentrations of phe-
nol results in nM and pM amounts of metabolite
in rats and fish, respectively, consideration must
be made as to the toxicity of metabolites relative
0
2
.0001) across temperatures between 11 °C (109
mM; 161935 pmol/min per mg) and 25 °C
(
3296 mM; 9409164 pmol/min per mg). As was
observed at 11 °C, the greatest capacity for HQ
production at 25 °C was measured with microso-
mal preparation c5, which was almost double
that of the lowest capacities measured for c1
and c2.
To better evaluate the differences in microso-
mal preparations that might explain noted differ-
ences in metabolism, comparisons were made of
individual fish sex, body weight and liver weight
for the three fish from which livers were pooled
for each microsomal preparation. This informa-
tion is presented in Table 3 along with protein,
P450 content, and EROD activities. Each prepa-
ration, with the exception of c3, contained hepa-
tocellular protein from both male and female fish.
The ratio of liver weight to body weight, or
hepatosomatic index (HST), was B1.3 for all fish
in the three preparations yielding the lower appar-
ent maximal rates of HQ production at either
temperature, c1, c2, c4. Both preparations
c3 and c5 included two fish with HSI\1.3,
and one trout in each with a HSI of 1.6. The HSI
can be used as an indication of enlarging liver
compared with body weight as typically seen in
fish such as trout as they seasonally approach
sexual maturity (Forlin and Haux, 1990). The two
fish with HSI of 1.6 were also the largest of the
entire data set in terms of body weight. The
measured EROD activities, an indicator of
metabolic O-dealkylation, were also elevated for
these preparations, as were the apparent Vmax
for HQ production at both 11 and 25 °C (Tables
1
and 2), although P450 protein did not appear
elevated in preparation c5.
4. Discussion
The microsomal biotransformation of phenol
was measured in the present study for the purpose
of obtaining specific metabolic rate information
needed for refinement of physiologically-based ki-
netic models for fish, and to obtain metabolic
pathway information needed for development and

R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
85

8
6
R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
to substrate. Specifically, HQ with a 96 h LC50 for
rainbow trout reported as 0.097 mg/l was shown
to be about 100 times more toxic than phenol
with an LC50 of 8.9 mg/l (DeGraeve et al., 1980).
Oxidation of HQ to BQ in vivo results in a
metabolite thought to be responsible for macro-
molecular binding and increased toxicity in mam-
malian systems (Schlosser et al., 1990). Secondary
metabolism of dihydroxybenzenes to quinones af-
ter trout microsomal biotransformation of 4-
methoxyphenol to HQ and 4-methoxycatechol
was presumed upon measurement of either BQ or
Nagel and Urich, 1983). However, in vitro work
with subcellular fractions have often utilized simi-
lar mM concentrations (Schlenk and Buhler 1991;
Dady et al., 1991; Soldano et al., 1992; Kolanczyk
et al., 1999). The apparent Km and Vmax deter-
mined for HQ and CAT formation are necessary
inputs for subsequent development of models to
predict phenol metabolism in vivo, and will be
compared with HQ production measured during
phenol inhalation exposures (McKim et al., 1999)
and in situ liver microdialysis delivery exposures
(
unpublished results).
4-methoxybenzoquinone (MBQ) (Kolanczyk, et
The current study applied a sensitive analytical
al., 1999). Definitive identification of either BQ or
MBQ was not possible due to co-elution of the
two compounds under the chromatographic con-
ditions. However, BQ was not detected in the
present study of phenol biotransformation. This
was likely due to the presence of high concentra-
tions of reducing equivalents in the microsomal
system, as well as the high reactivity of BQ
method (HPLC/ECD) to achieve an approxi-
mately 1000-fold increase in sensitivity over con-
ventional UV or colorimetric detection methods,
allowing the direct quantification of HQ and CAT
at low, yet toxicologically significant concentra-
tions observed in fish, thus allowing the calcula-
tion of rate constants for each individual
oxidative metabolite. The direct measurement of
HQ was only possible following the development
of these sensitive analytical methods due to the
small quantities produced by fish. Early studies of
phenol metabolism in fish detected sulfate and
glucuronide conjugates of both phenol and HQ
(
O’Brien, 1991). To substantiate this hypothesis,
experiments were performed in which various
concentrations of HQ and BQ were added to the
incubation matrix of microsomes and cofactor
regenerating system. Following a 15 min incuba-
tion, greater than 90% of added HQ was recov-
ered; however, the added BQ was not directly
detectable but measured as an observed 10–30%
conversion to HQ.
(
Kobayashi and Akitake, 1975; Layiwola and
Linnecar, 1981; Nagel, 1983; Nagel and Urich,
983; Kasokat, et al., 1987), but did not allow
1
direct measurement of the major metabolite. Us-
ing the technique applied to the current work,
McKim et al. (1999) reported the presence of HQ
in the urine and plasma of rainbow trout exposed
aqueously to phenol. Microsomal incubations
with phenol resulted in the measurement of uM
concentrations of HQ which were comparable to
the measured in vivo concentration of HQ in the
blood and urine of rainbow trout (McKim et al.,
The ranges of phenol concentrations used in the
present investigation were necessary to achieve
enzyme saturation at the temperatures under in-
vestigation, thus allowing the calculation of ki-
netic constants under standardized conditions. In
fact, the highest mM phenol concentrations used
were purposefully incorporated to determine if a
point was reached at which enzymatic rate was
adversely affected due to apparent protein toxic-
ity/denaturation or temperature dependent solu-
bility effects, thus assuring that lower phenol
concentrations were not adversely impacting
metabolic rate determinations. The in vitro con-
centrations utilized typically exceeded phenol con-
centrations administered by inhalation, skin
absorption, or ip injection in aquatic in vivo
studies (Nagel, 1983; Layiwola and Linnecar,
1
999). The present study extends the work of
McKim et al. (1999) through direct measurement
of metabolic rate and estimates of apparent Km
and Vmax of HQ and CAT from phenol in trout
at physiological temperature. Availability of this
type of information allows prediction of metabo-
lite formation needed for independent parameteri-
zation of kinetic models (Clewell and Andersen,
1985; Schlosser et al., 1993; Medinsky, 1995).
1981; McKim et al., 1999; Kasokat et al., 1987;

R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
87
The linear rate of microsomal ring-hydroxyla-
tion of phenol to CAT (1795 pmol/min per mg
per mM substrate) at 25 °C was similar to the
conversion of 4-methoxyphenol to 4-methoxycate-
chol (1791 pmol/min per mg per mM substrate)
observed by Kolanczyk et al. (1999). However, at
tion to affect measured rates. Therefore, a lower
temperature was used to allow calculation of Q10
values, which could be compared with those for
mammalian species.
Temperature effects on enzyme activity can be
described by Q , with a Q of two representing a
10
10
1
1 °C the rate of ortho-hydroxylation of phenol
doubling of enzymatic activity with a 10 °C tem-
perature rise. Values of Q10 around 2–2.5 have
been observed for O-dealkylation, ring-hydroxy-
(
791 pmol/min per mg per mM substrate), was
significantly less than the formation of 4-
methoxycatechol (1993 pmol/min per mg per
lation,
and
UDP-glucuronyltransferase
mM
substrate)
from
4-methoxyphenol
(Koivusaari and Andersson, 1984) and N-hydrox-
ylation (Dady et al., 1991) in rainbow trout. The
Q10 calculated using Vmax for production of HQ
was 2.3 and for CAT was 3.5, indicating a greater
effect of temperature on ortho-hydroxylation. The
increase in apparent Km with increasing tempera-
ture for both HQ and CAT production reported
here is consistent with previous observations
(Koivusaari and Andersson, 1984; Somero, 1978).
The biotransformation of phenol to HQ has been
measured in mice at 37 °C with reported Km and
Vmax of 0.38 mM and 7100 pmol/min per mg
protein, respectively (Lunte and Kissinger, 1983).
A Q10 based on trout HQ production rate at
11 °C and mouse HQ production at 37 °C would
be 2.7, close to frequently observed Q10 values of
2–2.5 perhaps suggesting similarity in maximum
capacity of enzymes responsible for para-hydrox-
ylation of phenol to HQ between these species.
However, the Km measured at the physiologi-
cal temperature for each species is much lower in
mice than trout (ca. a 40-fold difference), indicat-
ing mice enzymes exhibit a greater affinity for the
substrate than trout. Lunte and Kissinger (1983)
also observed formation of CAT from phenol in
mouse microsomes but did not quantify its
occurrence.
It should be noted that the data presented here
showed some apparent deviation from Michaelis–
Menten kinetics, especially at lower phenol con-
centration at 25 °C (Fig. 3A and B). These
deviations from the curve fit to the data (Fig. 3A
and B) could be explained by either an enzyme lag
effect or quantification uncertainties at relatively
low HQ and CAT concentrations near their detec-
tion limits. Even though there are apparent devia-
tions from Michaelis–Menten kinetics, the Km
and Vmax parameters are good descriptors com-
(
Kolanczyk et al., 1999). Further analysis of the
linear rate of HQ formation from para-hydroxyla-
tion of phenol at 11 °C (2296 pmol/min per mg
per mM substrate) and 25 °C (45917 pmol/min
per mg per mM substrate) was nearly the same as
that from the O-dealkylation of 4-MP (2292
pmol/min per mg per mM substrate and 3491
pmol/min per mg per mM substrate, respectively),
(
Kolanczyk et al., 1999). Determination of the
rate constants through direct measurements of
low pmol formation rates allows comparison of
specific rate data not previously obtainable in
aquatic species, as well as comparisons of forma-
tion rates of similar products (i.e. HQ) derived
from different enzymatic processes (O-dealkyla-
tion vs. ring-hydroxylation). This type of informa-
tion is essential to the development of accurate
predictive models of biotransformation and toxic-
ity in aquatic species.
The present study provides Michaelis–Menten
rate constants at 11 and 25 °C for future species
comparison and extrapolation. The two tempera-
tures provide a basis for rate comparison to other
‘
cold-water’ species (brook trout, lake trout) typi-
cally run at 11 °C (work in progress at this
laboratory) and ‘warm-water’ species (medaka,
catfish, etc.) typically run at 25 °C. In the past,
much in vitro fish metabolic rate data was col-
lected at 25 °C to maximize enzymatic rates.
While not directly applicable to this study, the
collection of rate data at 25 °C will allow com-
parison to be made across reaction types, as well
as species. Rates determined at physiological tem-
perature, 11 °C, are also important for in vitro to
in vivo extrapolation. Measurements made at
37 °C are not appropriate for cold water fish
species with a high potential for protein denatura-

8
8
R.C. Kolanczyk, P.K. Schmieder / Toxicology 176 (2002) 77–90
monly used for approximating maximal reaction
rates. Therefore, these statistics are still deemed
appropriate for comparison of species and other
substrates as well as in vitro to in vivo extrapola-
tion. To justify any use of a new descriptor other
than constants used to describe Michaelis–
Menten type kinetics would require a more rigor-
ous data set than available here.
tion of metabolites at low picomolar
concentrations present in aquatic species was
demonstrated. The availability of this type of
information is essential to the development of
accurate models predictive of increased toxicity
due to metabolic activation.
Acknowledgements
The current study, and that of Kolanczyk et al.
(
1999), provide pathway and rate data for
The authors would like to acknowledge the
review of the manuscript by James M. McKim
III, Douglas W. Kuehl, Russell J. Erickson, and
Jose A. Serrano. The information in this docu-
ment has been funded wholly (or in part) by the
US Environmental Protection Agency. It has been
subjected to review by the National Health and
Environmental Effects Research Laboratory and
approved for publication. Approval does not sig-
nify that the contents reflect the views of the
Agency, nor does mention of trade names or
commercial products constitute endorsement or
recommendation for use.
metabolic production of HQ in rainbow trout
from two different enzymatic reaction types, ring-
hydroxylation and O-dealkylation. Trout cells
have also been shown to exhibit biochemical re-
sponses indicative of quinone toxicity that are
qualitatively similar to those reported for mam-
malian cells (Tapper et al., 2000), although direct
comparison of concentration–response profiles
are somewhat complicated by differences in cellu-
lar media and physiological temperatures, and
paucity of measured exposure concentrations.
Therefore, pathways and rates of metabolic con-
versions resulting in production of HQ and qui-
none species are important to quantify in fish, as
well as in mammals, when attempting to under-
stand and predict reactive chemical toxicities
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